Antibodies for detection of mycoplasma hyopneumoniae and methods of making and using same

ABSTRACT

Antibodies that detect M. hyopneumoniae, methods of making those antibodies, and methods of using those antibodies including, for example, in a diagnostic immunoassay, are described. Such a diagnostic assay may be used in pen-side testing for detection of M. hyopneumoniae.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 63/243,818, filed Sep. 14, 2021, which is incorporatedherein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under 2020-67021-31956awarded by the National Institute of Food and Agriculture, USDA. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted tothe United States Patent and Trademark Office via Patent Center as anXML file entitled “0110-000665US01” having a size of 10 kilobytes andcreated on Sep. 13, 2022. Due to the electronic filing of the SequenceListing, the electronically submitted Sequence Listing serves as boththe paper copy required by 37 CFR § 1.821(c) and the CRF required by §1.821(e). The information contained in the Sequence Listing isincorporated by reference herein.

SUMMARY

This disclosure describes antibodies that detect M. hyopneumoniae. Thisdisclosure further describes methods of making those antibodies and ofusing those antibodies including, for example, in a diagnosticimmunoassay. Such a diagnostic assay may be used in pen-side testing fordetection of M. hyopneumoniae.

In one aspect, the antibody includes one or more complementarydetermining regions (CDRs) of the heavy chain variable region of MAb2C9.B3, one or more CDR of the light chain variable region of MAb2C9.B3, or both one or more complementary determining regions (CDRs) ofthe heavy chain variable region of MAb 2C9.B3 and or more CDR of thelight chain variable region of MAb 2C9.B3.

In another aspect, the antibody includes one or more complementarydetermining regions (CDRs) of the heavy chain variable region of4G11.A3, one or more CDR of the light chain variable region of 4G11.A3,or both one or more complementary determining regions (CDRs) of theheavy chain variable region of MAb 4G11.A3 and or more CDR of the lightchain variable region of MAb 4G11.A3.

In one or more embodiments of either aspect, the antibody is humanized.

In one or more embodiments of either aspect, the antibody is an antibodyfragment.

In one or more embodiments of either aspect, the antibody is an IgGantibody.

In one or more embodiments of either aspect, the antibody is amonoclonal antibody.

In one or more embodiments of either aspect, the antibody is a chimericantibody.

In another aspect, this disclosure describes a composition that includesan antibody that detects M. hyopneumoniae.

In another aspect, this disclosure describes a kit that includes anantibody that detects M. hyopneumoniae.

In another aspect, this disclosure describes a method that includesusing the antibody that detects M. hyopneumoniae in an in vitro or invivo diagnostic or therapeutic method.

In one or more embodiments, the method includes detecting the presenceof M. hyopneumoniae in a subject.

In one or more embodiments, the method includes detecting of M.hyopneumoniae in a sample obtained from a subject.

In one or more embodiments, the method includes performing a magneticbioassay or enzyme-linked immunosorbent assay (ELISA).

In one or more embodiments, the method includes detecting a change inthe magnetoresistance ratio (AMR) from a giant magnetoresistance (GMR)sensor.

The above summary is not intended to describe each disclosed embodimentor every implementation of the present invention. The description thatfollows more particularly exemplifies illustrative embodiments. Inseveral places throughout the application, guidance is provided throughlists of examples, which examples can be used in various combinations.In each instance, the recited list serves only as a representative groupand should not be interpreted as an exclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of a monoclonal antibody development process byhybridoma. Hybridoma cells formed by fusion of antibody-producing cellsand myeloma cells are selected by HAT medium. These hybrid cells arescreened by ELISA to determine specific antibody secreting clones andsub-cloned by limiting dilution.

FIG. 2 . Characterization of monoclonal antibodies to M. hyopneumoniae.Data shows reactivity of selected monoclonal antibodies to M.hyopneumoniae, M. hyorhinis, and M. flocculare by ELISA. Microtiterwells were coated with whole cell lysate of indicated Mycoplasma speciesand detected using culture supernatant from different hybridoma clones(prior to subcloning) followed by goat anti-mouse IgG-HRP and TMB/H202substrate. Pre-immune mouse sera was included as a control. Blank didnot include any antibody, only a sample diluent (phosphate buffer saline(PBS) with 1% bovine serum albumin (BSA)) used to dilute the samples.Dotted line indicates the cut-off value.

FIG. 3 . Characterization of monoclonal antibodies to M. hyopneumoniae.Specific reactivity of two monoclonal antibodies, MAb 2C9.B3 and MAb4G11.A3, to M. hyopneumoniae but not to M. hyorhinis and M. flocculareas determined by Western blot. Whole cell lysate of (1) M. hyopneumoniae(2) M. hyorhinis and (3) M. flocculare were run on 12.5% SDS-PAGE,transferred to PVDF membrane and reacted with MAb 2C9.B3 or MAb 4G11.A3,followed by goat anti-mouse IgG-HRP conjugate. (A) Results obtained witha DAB/H202 substrate. (B) Results obtained with a chemiluminescentsubstrate. M: Protein size marker. MAb 2C9.B3 binds to an ˜72 kDaprotein and MAb 4G11.A3 binds to an ˜45 kDa protein of M. hyopneumoniae.

FIG. 4 . Results of an exemplary sandwich ELISA using rabbit anti-M.hyopneumoniae as capture antibody and MAb 2C9.B3 (also referred toherein as MAb 2) to M. hyopneumoniae as detection antibody for detectionof multiple isolates of M. hyopneumoniae from different swine farms.Dotted line indicates the cut-off value.

FIG. 5 . Results of an exemplary sandwich ELISA using rabbit anti-M.hyopneumoniae as capture antibody and MAb 4G11.A3 (also referred toherein as MAb 4) to M. hyopneumoniae as detection antibody for detectionof multiple isolates of M. hyopneumoniae from different swine farms.Dotted line indicates the cut-off value.

FIG. 6 . Exemplary results of M. hyopneumoniae detection as described inExample 2. MAb2C9.B3 versus MAb4G11.A3 with background Mycoplasma.Results of an exemplary sandwich ELISA assay, as further described inExample 2. FIG.

FIG. 7 shows a portable hand-held giant magnetoresistance (GMR)diagnostic platform and exemplary schematic and results of a magneticsandwich assay that could be used for M. hyopneumoniae detection. (A)Real-time data collection and data transmission may either be donewirelessly thorough Bluetooth to a smartphone, a tablet, or a laptop orthrough USB connection to a desktop computer. (B) Exemplary componentsof hand-held detection platform. (C) An exemplary fabricated GMR chip.(D) A schematic representation of a magnetic sandwich assay, MACSindicates an exemplary magnetic nanoparticle, available from MiltenyiBiotech (Bergisch Gladbach, Germany). (E) Real-time binding curves fortargeted binding of different concentrations of target. In thisexemplary example, binding curves for influenza A virus (IAV) H3N2v areshown. (F) Average signal from different concentrations of IAV H3N2v at10 minutes after sample addition.

FIG. 8 . Specific reactivity of monoclonal antibody 4G11.A3 to M.hyopneumoniae determined by sandwich ELISA. Microtiter wells were coatedwith purified Mab 4G11.A3. Whole cell lysate of indicated Mycoplasmaspecies or other common swine respiratory pathogens—Streptococcus suis,Pasturella multocida, Porcine reproductive and respiratory syndromevirus (PRRSv) isolate VR2332, Influenza A virus (IAV) H1N1, and IAV H3N2were allowed to react with coated Mab 4G11.A3. Bound proteins weredetected using rabbit polyclonal antibody to M. hyopneumoniae followedby goat anti-rabbit IgG-HRP conjugate and TMB/H₂O₂ substrate. Dottedline indicates the cut off value. p FIG. 9 . Concentration curve ofMycoplasma hyopneumoniae detection by sandwich ELISA using MAb4G11.A3 ascapture antibody. Microtiter wells were coated with purified Mab4G11.A3. Whole cell lysate of M. hyopneumoniae was serially dilutedtwo-fold in PBS and added to the wells. After washing, bound proteinswere detected using rabbit polyclonal antibody to M. hyopneumoniaefollowed by goat anti-rabbit IgG-HRP conjugate and TMB/H202 substrate.Dotted line indicates the cut off value.

FIG. 10 . Concentration curve of M. hyopneumoniae detection in nasalswab sample by sandwich ELISA using MAb4G11.A3 as capture antibody.Microtiter wells were coated with purified Mab 4G11.A3. Whole celllysate of Mycoplasma hyopneumoniae was serially diluted two-fold inpooled nasal swab sample of pigs negative for M hyopneumoniae and addedto the wells. After washing, bound proteins were detected using rabbitpolyclonal antibody to M. hyopneumoniae followed by goat anti-rabbitIgG-HRP conjugate and TMB/H₂O₂ substrate. Dotted line indicates the cutoff value.

FIG. 11 . Sandwich ELISA using MAb4G11.A3 detected M. hyopneumoniae intracheal swab of experimentally infected pigs. Pigs were experimentallyinfected with M. hyopneumoniae. At 0 day post infection (dpi) and 28dpi, tracheal swab was collected and tested using ELISA.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This disclosure describes antibodies that bind to M. hyopneumoniae. Thisdisclosure further describes methods of making those antibodies and ofusing those antibodies including, for example, in a diagnosticimmunoassay. Such a diagnostic assay may be used in pen-side testing fordetection of M. hyopneumoniae.

As further described herein, the antibodies that bind to M.hyopneumoniae may be used for the detection of M. hyopneumoniae. Usingantibodies that bind to M. hyopneumoniae is more accurate than existingdiagnostic assays for M. hyopneumoniae that detect serum antibodies.Moreover, using antibodies that bind to M. hyopneumoniae (particularlyin the context of, for example, a kit) is easier than existingdiagnostic assays for M. hyopneumoniae that use PCR to detect thebacterial genome; in contrast to PCR, a kit that include antibodies thatbind to M. hyopneumoniae requires no specialized expertise.

Antibodies

In one aspect, this disclosure describes antibodies that bind to M.hyopneumoniae. As used herein, the term “antibody” refers generally animmunoglobulin or a fragment thereof. Thus, as used herein, the term“antibody” encompasses not only immunoglobulins with an intact Fcregion, but also antibody fragments capable of binding to a biologicalmolecule (such as an antigen or receptor) or a portion thereof,including but not limited to Fab, Fab′, F(ab′)₂, pFc′, Fd, Fd′, Fv, dAB,a single domain antibody (sdAb), a variable fragment (Fv), asingle-chain variable fragment (scFv) or a disulfide-linked Fv (sdFv), adiabody or a bivalent diabody, a linear antibody, a single-chainantibody molecule, or a multispecific antibody (e.g., a tribody) formedfrom antibody fragments. The antibody can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgAland IgA2), or subclass.

In one or more embodiments, the antibody can be a humanized antibodyderived from an animal single domain antibody. While an scFv has a heavyvariable chain component and a light variable chain component joined bya flanking sequence, a single domain antibody consists of a singlemonomeric variable chain—i.e., a variable heavy chin or a variable lightchain—that is capable of specifically engaging a target. A single domainantibody may be derived from an antibody of any suitable animal such as,for example, a camelid (e.g., a llama or camel) or a cartilaginous fish(e.g., a wobbegong or a nurse shark). A single domain antibody canprovide superior physical stability, an ability to bind deep grooves,and increased production yields compared to larger antibody fragments.

In one or more embodiments, an antibody that binds to M. hyopneumoniaeis a monoclonal antibody. In one or more embodiments, antibodies thatbind to M. hyopneumoniae include monoclonal antibodies produced by thehybridoma cell lines (also referred to herein as clones) 2C9.B3 or4G11.A3; such antibodies are also referred to herein as MAb 2C9.B3 (orMAb2) and MAb 4G11.A3 (or MAb4), respectively.

In one or more embodiments, an antibody that binds to M. hyopneumoniaepreferably does not bind to other commensal or pathogenic species ofswine Mycoplasma. For example, in one or more embodiments, an antibodythat binds to M. hyopneumoniae preferably does not bind to M. hyorhinisor M. flocculare.

In one or more embodiments, the antibody is an isolated antibody. In oneor more embodiments, the antibodies may be isolated or purified byconventional immunoglobulin purification procedures, such as proteinA-Sepharose chromatography, protein G-Sepharose chromatography,hydroxyapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

In one or more embodiments, an antibody that binds to M. hyopneumoniaemay include a derivative of an antibody that is modified or conjugatedby the covalent attachment of any type of molecule to the antibody. Suchantibody derivatives include, for example, antibodies that have beenmodified by glycosylation, acetylation, PEGylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, toxins, or linkage to a cellular ligand or otherprotein. Any of numerous chemical modifications may be carried out byknown techniques, including, but not limited to, specific chemicalcleavage, acetylation, formylation, and metabolic synthesis oftunicamycin. Additionally, the derivatives may contain one or morenon-classical amino acids.

An antibody that binds to M. hyopneumoniae may be coupled directly orindirectly to a detectable marker by techniques well known in the art. Adetectable marker is an agent detectable using, for example,spectroscopic, photochemical, biochemical, immunochemical, or chemicalmethods. Useful detectable markers include, but are not limited to,magnetic nanoparticles, magnetic microbeads, fluorescent dyes andmaterials, chemiluminescent compounds and materials, bioluminescentmaterials, electron-dense reagents, enzymes, coenzymes, coloredparticles, biotin, digoxigenin, or radioactive materials that include aradioisotope. A detectable marker may generate a measurable signal, suchas radioactivity, fluorescent light, color, or enzyme activity.Antibodies conjugated to detectable markers may be used for diagnosticor therapeutic purposes. The detectable marker may be coupled orconjugated either directly to the antibody or indirectly, through anintermediate such as, for example, a linker known in the art, usingtechniques known in the art. See, for example, U.S. Pat. No. 4,741,900,describing the conjugation of metal ions to antibodies for diagnosticuse. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, and acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride and phycoerythrin; an example of a luminescent materialincludes luminol; examples of bioluminescent materials includeluciferin, and aequorin; and examples of suitable radioactive materialinclude iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹¹In, ¹¹²In, ¹¹³mIn, ¹¹⁵mIn), technetium (⁹⁹Tc,⁹⁹mTc), thallium (²⁰¹Ti) gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd),molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd,¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ₉₀Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, and⁹⁷Ru. Techniques for conjugating such moieties to antibodies arewell-known.

In an exemplary embodiment, the antibody may be conjugated to biotin. Inanother exemplary embodiment, the antibody may be directly or indirectlyconjugated to a magnetic nanoparticle, such as MACS, streptavidin-coatedsuperparamagnetic microbeads, available from Miltenyi Biotech (BergischGladbach, Germany) and Miltenyi Biotec, Inc. (Auburn, Calif.). Forexample, as shown in FIG. 7D, the antibody may be conjugated to biotinwhich allows binding to a streptavidin-conjugated magnetic microbead.

Also included in the present disclosure are monoclonal antibodiesproduced by progeny or derivatives of these hybridoma cell lines,monoclonal antibodies produced by equivalent or similar hybridoma celllines, and/or recombinant derivatives made thereof. In one or moreembodiments, an antibody that binds to M. hyopneumoniae includes arecombinantly derived monoclonal that includes one or morecomplementarity determining regions (CDRs) of MAb 2C9.B3 or MAb 4G11.A3.

An intact antibody molecule has two heavy (H) chain variable regions(abbreviated herein as V_(H)) and two light (L) chain variable regions(abbreviated herein as V_(L)). The V_(H) and V_(L) regions may befurther subdivided into regions of hypervariability, termed“complementarity determining regions” (“CDRs”), interspersed withregions that are more conserved, termed “framework regions” (“FRs”). Theextent of the FRs and CDRs has been precisely defined (see, Kabat et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, and Chothia et al. (1987) J. Mol. Biol. 196: 901-917). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

In one or more embodiments, an antibody that binds to M. hyopneumoniaeincludes the V_(H) domain of the monoclonal antibody produced byhybridoma cell line 2C9.B3 (MAb 2C9.B3; SEQ ID NO:2) or the V_(H) domainof the monoclonal antibody produced by hybridoma cell line 4G11.A3 (MAb4G11.A3; SEQ ID NO:6). In one or more embodiments, an antibody thatbinds to M. hyopneumoniae includes the V_(L) domain of the monoclonalantibody produced by hybridoma cell line 2C9.B3 (MAb 2C9.B3; SEQ IDNO:4) or the V_(L) domain of the monoclonal antibody produced byhybridoma cell line 4G11.A3 (MAb 4G11.A3; SEQ ID NO:8). In one or moreembodiments, an antibody that binds to M. hyopneumoniae includes theV_(H) domain and the V_(L) domain of MAb 2C9.B3 or the V_(H) domain andthe V_(L) domain of MAb 4G11.A3. In one or more embodiments, an antibodythat binds to M. hyopneumoniae may contain one, two, three, four, five,six, or more amino acid substitutions compared to the amino acidsequences of the V_(H) domains and/or the V_(L) domains identified abovewherein the amino acid substitutions do not substantially affect bindingof the antibody to M. hyopneumoniae. The amino acid substitutions mayoccur in the FRs or the CDRs.

In one or more embodiments, an antibody that binds to M. hyopneumoniaeincludes at least one CDR of the V_(H) domain of MAb 2C9.B3 (amino acids50-54 of SEQ ID NO:2, amino acids 69-84 of SEQ ID NO:2, or amino acids117-126 of SEQ ID NO:2) or at least one CDR of the V_(H) domain of MAb4G11.A3 (amino acids 50-54 of SEQ ID NO:6, amino acids 69-85 of SEQ IDNO:6, or amino acids 118-132 of SEQ ID NO:6). In one or moreembodiments, an antibody that binds to M. hyopneumoniae includes atleast two CDRs of the V_(H) domain of MAb 2C9.B3 or at least two CDRs ofthe V_(H) domain of MAb 4G11.A3. In one or more embodiments, an antibodythat binds to M. hyopneumoniae includes all three CDRs of the V_(H)domain of MAb 2C9.B3 or all three CDRs of the V_(H) domain of MAb4G11.A3.

Additionally or alternatively, in one or more embodiments, an antibodythat binds to M. hyopneumoniae includes at least one CDR of the V_(L)domain of MAb 2C9.B3 (amino acids 44-54 of SEQ ID NO:4, amino acids70-76 of SEQ ID NO:4, or amino acids 109-117 of SEQ ID NO:4) or at leastone CDR of the V_(L) domain of MAb 4G11.A3 (amino acids 46-55 of SEQ IDNO:8, amino acids 71-77 of SEQ ID NO:8, or amino acids 110-118 of SEQ IDNO:8). In one or more embodiments, an antibody that binds to M.hyopneumoniae includes at least two CDRs of the V_(L) domain of MAb2C9.B3 or at least two CDRs of the V_(L) domain of MAb 4G11.A3. In oneor more embodiments, an antibody that binds to M. an antibody that bindsto M. hyopneumoniae

In one or more embodiments, may contain one, two, three, four, five,six, or more amino acid substitutions in one or more CDRs identifiedabove that do not substantially affect binding of the antibody to M.hyopneumoniae.

In one or more embodiments, an antibody that binds to M. hyopneumoniaemay contain one, two, three, four, five, six, or more amino acidsubstitutions in one or more framework regions (FRs). In one or moreembodiments, the substitution or substitutions in the FRs do notsubstantially affect binding of the antibody to M. hyopneumoniae.

In one or more embodiments, an antibody that binds to M. hyopneumoniaeincludes an amino acid sequence having at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity to an amino acid sequence of atleast one CDR of a V_(H) domain of MAb 2C9.B3 or MAb 4G11.A3.

In one or more embodiments, an antibody that binds to M. hyopneumoniaeincludes an amino acid sequence having at least 70%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to an amino acid sequence of atleast one CDR of a V_(L) domain of MAb 2C9.B3 or MAb 4G11.A3.

In another aspect, this disclosure describes an isolated nucleic acidsequence that encodes any embodiment of an antibody that binds to M.hyopneumoniae (or fragment thereof). In one or more embodiments, theisolated nucleic acid encodes an antibody that includes one or more ofthe CDRs of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8. Giventhe amino acid sequence of any antibody or antibody fragment, a personof ordinary skill in the art can determine the full scope ofpolynucleotides that encode that amino acid sequence using conventional,routine methods. In one or more embodiments, the isolated nucleotide caninclude the portion or portions of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, or SEQ ID NO:7 that encode one or more CDRs of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, or SEQ ID NO:8.

As used herein, the term “nucleic acid” or “oligonucleotide” refers topolynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). Nucleic acids include but are not limited to genomic DNA, cDNA,mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced andchemically synthesized molecules such as aptamers, plasmids, anti-senseDNA strands, shRNA, ribozymes, nucleic acids conjugates, andoligonucleotides. A nucleic acid may be single-stranded,double-stranded, linear, or covalently circularly closed molecule. Anucleic acid can be isolated. The term “isolated nucleic acid” meansthat the nucleic acid (i) was amplified in vitro, for example viapolymerase chain reaction (PCR), (ii) was produced recombinantly bycloning, (iii) was purified, for example, by cleavage and separation bygel electrophoresis, (iv) was synthesized, for example, by chemicalsynthesis, or (vi) extracted from a sample. A nucleic might beintroduced—i.e., transfected—into cells. When RNA is used to transfectcells, the RNA may be modified by stabilizing modifications, capping, orpolyadenylation.

As used herein “amplified DNA” or “PCR product” refers to an amplifiedfragment of DNA of defined size. Various techniques are available andwell known in the art to detect PCR products. PCR product detectionmethods include, but are not restricted to, gel electrophoresis usingagarose or polyacrylamide gel and adding ethidium bromide staining (aDNA intercalant), labeled probes (radioactive or non-radioactive labels,southern blotting), labeled deoxyribonucleotides (for the directincorporation of radioactive or non-radioactive labels) or silverstaining for the direct visualization of the amplified PCR products;restriction endonuclease digestion, which relies on agarose gelelectrophoresis, polyacrylamide gel electrophoresis, or high-performanceliquid chromatography (HPLC); dot blots, using the hybridization of theamplified DNA on specific labeled probes (radioactive or non-radioactivelabels); high-pressure liquid chromatography using ultravioletdetection; electro-chemiluminescence coupled with voltage-initiatedchemical reaction/photon detection; and direct sequencing usingradioactive or fluorescently labeled deoxyribonucleotides for thedetermination of the precise order of nucleotides with a DNA fragment ofinterest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing,fluorescence, gel electrophoresis, magnetic beads, allele specificprimer extension (ASPE) and/or direct hybridization.

Generally, nucleic acid can be extracted, isolated, amplified, oranalyzed by a variety of techniques well-established and known to thoseof skill in the art. Examples of nucleic acid analysis include, but arenot limited to, sequencing and DNA-protein interaction. Sequencing maybe by any method known in the art. DNA sequencing techniques includeclassic dideoxy sequencing reactions (Sanger method) using labeledterminators or primers and gel separation in slab or capillary, and nextgeneration sequencing methods such as sequencing by synthesis usingreversibly terminated labeled nucleotides, pyrosequencing, 454sequencing, Illumina/Solexa sequencing, allele specific hybridization toa library of labeled oligonucleotide probes, sequencing by synthesisusing allele specific hybridization to a library of labeled clones thatis followed by ligation, real time monitoring of the incorporation oflabeled nucleotides during a polymerization step, polony sequencing, andSOLiD sequencing. Separated molecules may be sequenced by sequential orsingle extension reactions using polymerases or ligases as well as bysingle or sequential differential hybridizations with libraries ofprobes.

In another aspect, this disclosure describes a host cell including anyof the isolated nucleic acid sequences and/or proteins described herein.Thus, this disclosure encompasses translation of a nucleic acid (e.g.,an mRNA) by a host cell to produce an antibody encoded by the nucleicacid.

The nucleic acid constructs described herein may be introduced into ahost cell, thus allowing expression of the antibody within the cell,thereby generating a genetically engineered cell. A variety of methodsare known in the art and suitable for introducing a nucleic acid into acell, including viral and non-viral mediated techniques. Examples oftypical non-viral mediated techniques include, but are not limited to,electroporation, calcium phosphate mediated transfer, nucleofection,sonoporation, heat shock, magnetofection, liposome mediated transfer,microinjection, microproj ectile mediated transfer (nanoparticles),cationic polymer mediated transfer (DEAE-dextran, polyethylenimine,polyethylene glycol (PEG) and the like) or cell fusion. Other methods oftransfection include proprietary transfection reagents such asLIPOFECTAMINE (Thermo Fisher Scientific, Inc., Waltham, Mass.), HILYMAX(Dojindo Molecular Technologies, Inc., Rockville, Md.), FUGENE (PromegaCorp., Madison, Wis.), JETPEI (Polyplus Transfection, Illkirch, France),EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc.USA, San Diego, Calif.).

The nucleic acid constructs described herein may be introduced into ahost cell to be altered, thus allowing expression within the cell of theprotein encoded by the nucleic acid. A variety of host cells are knownin the art and suitable for protein expression. Examples of typical cellused for transfection and protein expression include, but are notlimited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insectcell, or a plant cell such as, for example, E. coli, Bacillus,Streptomyces, Pichia pastoris, Salmonella typhimurium, Drosophila S2,Spodoptera SJ9, CHO, COS (e.g., COS-7),3T3-F442A, HeLa, HUVEC, HUAEC,NIH 3T3, Jurkat, 293, 293H, or 293F.

The antibody may be an antibody from any suitable species. In one ormore embodiments, the antibody may be a mouse antibody. In one or moreembodiments, the antibody may be a rat antibody. In one or moreembodiments, the antibody may be a rabbit antibody.

In one or more embodiments, the antibody is an IgG antibody. In one ormore embodiments, the antibody may be an antibody or an IgG subclassincluding, for example, IgG1, IgG2, IgG3 or IgG4. In one or moreembodiments, the antibody may be a mouse IgG of one of the followingsub-classes: IgG1, IgG2A, IgG2B, IgG2C, and IgG3. In one or moreembodiments, the antibody may be a mouse IgG1.

In one or more embodiments, the antibody may include a kappa lightchain. In one or more embodiments, the antibody may include a lambdalight chain.

In one or more embodiments, the monoclonal antibody includes anantigen-binding fragment such as, for example, a Fab fragment, a Fab′fragment, an F(ab)₂ fragment, and/or an Fv fragment.

In one or more embodiments, the antibody may be a humanized antibody. Anantibody that binds to M hyopneumoniae may be humanized by any suitablemethod. Techniques for producing humanized monoclonal antibodies may befound, for example, in Jones et al. (1986) Nature 321:522 and Singer etal. (1993) J. Immunol. 150:2844. For example, humanization of theantibody may include changes to the antibody to reduce theimmunogenicity of the antibody when used in humans. In one or moreembodiments, a humanized antibody that binds to M. hyopneumoniae mayinclude at least a portion of an immunoglobulin constant region (Fc) ofa human immunoglobulin. A humanized antibody that binds to M.hyopneumoniae may include, in one or more embodiments, a humanimmunoglobulin (recipient antibody) in which residues from one or morecomplementary determining regions (CDRs) of the recipient antibody arereplaced by residues from one or more CDRs of a non-human speciesantibody (donor antibody), such as mouse, rat, or rabbit antibody, thatbinds to M. hyopneumoniae. In one or more embodiments, Fv frameworkresidues of a human immunoglobulin may be replaced by correspondingnon-human residues from an antibody that binds to M. hyopneumoniae.

In one or more embodiments, a monoclonal antibody includes a chimericantibody—i.e., an antibody in which different portions are derived fromdifferent animal species. A chimeric antibody may be obtained by anyknown method such as, for example, splicing the genes from a mouseantibody molecule with appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biologicalspecificity. Methods for preparing chimeric antibodies are establishedand well-known to those of ordinary skill in the art.

Hybridoma Cell Lines

This disclosure further describes hybridoma cell lines (also referred toherein as “clones” or “antibody clones”) 2C9 and 4G11, and theirsubclones 2C9.B3 and 4G11.A3 (also referred to by the internallaboratory designations M. hyo 2C9.B3 and M. hyo 4G11.A3). 2C9.B3 and4G11.A3 express monoclonal antibodies MAb 2C9.B3 and MAb 4G11.A3,respectively. In one or more embodiments, a monoclonal antibody producedby a hybridoma cell line binds to M. hyopneumoniae.

Hybridoma cell lines may be obtained by various techniques familiar tothose skilled in the art. In one or more embodiments, the animalimmunized to produce a hybridoma cell line is preferably a mammal. Inone or more embodiments, the immunized animal is a rat including (e.g.,a Wistar rat) or a mouse (e.g., a BALB/C mouse). In one or moreembodiments, the cells obtained from the immunized animal to produce ahybridoma are spleen cells. In one or more embodiments, the cellsobtained from the immunized animal to produce a hybridoma are preferablylymphocytes. In one or more embodiments, the hybridoma is produced usinga myeloma cell such as, for example, an SP2/O cell.

Other known methods of producing transformed B cell lines that producemonoclonal antibodies may also be used.

Methods of Making the Antibodies

In another aspect, this disclosure describes methods of makingantibodies that detect M. hyopneumoniae.

A monoclonal antibody may be obtained by any suitable technique.

In one or more embodiments, a monoclonal antibody that binds to M.hyopneumoniae may be produced by a hybridoma cell described herein.

In one or more embodiments, an antibody that binds to M. hyopneumoniaemay be made by recombinant DNA methods, produced by phage display,and/or produced by combinatorial methods. DNA encoding an antibody thatbinds to M. hyopneumoniae may be readily isolated and sequenced usingconventional procedures. In one or more embodiments, a hybridoma celldescribed herein may serve as a source of such DNA. Once isolated, theDNA may be transfected into a host cell (including, for example, simianCOS cells, Chinese hamster ovary (CHO) cells, human embryonic kidneycells (HEK), or myeloma cells that do not otherwise produceimmunoglobulin protein) or introduced into a host cell by genome editing(for example, using a CRISPR-Cas system) to obtain the synthesis ofantibodies in a recombinant host cell. The DNA encoding an antibody thatbinds to M. hyopneumoniae may be modified to, for example, humanize theantibody.

Methods of Using the Antibodies

In yet another aspect, this disclosure describes methods of usingantibodies that bind to M. hyopneumoniae. An antibody that binds to M.hyopneumoniae, as described herein, may be used for any suitableapplication. For example, a monoclonal antibody may be used in both invitro and in vivo diagnostic and therapeutic methods. In one or moreembodiments, the diagnostic methods may preferably be in vitrodiagnostic methods including, for example, detecting M. hyopneumoniae ina sample from a subject.

In one or more embodiments, an antibody may be used to label and/ordetect M. hyopneumoniae, in vivo or in vitro.

In one or more embodiments, an antibody may be used to detect thepresence or absence of M. hyopneumoniae in a sample from a subject. Inone or more embodiments, detecting the presence of M. hyopneumoniae mayinclude identifying an amount of M. hyopneumoniae in a sample from asubject.

As used herein, the term “subject” includes, but is not limited to,humans and non-human vertebrates. Non-human vertebrates includelivestock animals, companion animals, and laboratory animals. Non-humansubjects also include non-human primates as well as rodents, such as,but not limited to, a rat or a mouse. Non-human subjects also include,without limitation, chickens, horses, cows, pigs, goats, dogs, cats,guinea pigs, hamsters, mink, and rabbits. In an exemplary embodiment,the subject is a pig.

Any suitable sample obtained from the subject may be used. Exemplarysamples include a blood sample, a tissue sample, a laryngeal swab, anasal swab, a bronchial swab, a sample obtained by deep trachealcatheter, etc. In one or more embodiments, a sample may include atracheal fluid and/or another respiratory tract fluid. In one or moreembodiments, a sample may preferably include a tracheal fluid. When thesample includes a tissue sample, the antibody that binds to M.hyopneumoniae may be used to detect M hyopneumoniae in situ.

In one or more embodiments, detecting the presence of M. hyopneumoniaemay include performing an assay on a sample from a subject. Exemplaryassays include a magnetic bioassay, an enzyme-linked immunosorbent assay(ELISA), Western blot, immunohistochemistry, immunocytochemistry, flowcytometry, immunoprecipitation, etc. ELISA may include direct ELISA,indirect ELISA, and sandwich ELISA.

In one or more embodiments, detecting the presence of M. hyopneumoniaein a sample from a subject may be used to diagnose a subject with a M.hyopneumoniae infection.

This disclosure further described a kit or a device including anantibody. For example, the kit or device may include a composition thatincludes an anti-M. hyopneumoniae monoclonal antibody. The antibodies inthe kit or device may be labeled with one or more detectable markers, asdescribed herein. In one or more embodiments, the device may include agiant magnetoresistance (GMR)-based diagnostic immunoassay platform. Inone or more embodiments, the device may preferably be portable (that is,easily conducted outside of a laboratory), allowing for, for example,pen-side testing for M. hyopneumoniae.

In an exemplary embodiment, an antibody that binds M. hyopneumoniae maybe used in a diagnostic immunoassay. Such an immunoassay may be used inpen-side testing for detection of M. hyopneumoniae. Such an immunoassaymay preferably be portable.

As further described in Example 4, the immunoassay may include amagnetic bioassay and, more specifically, a giant magnetoresistance(GMR)-based diagnostic immunoassay platform. Such a platform isdescribed for the detection of Influenza A in Wu et al., ACS Sens. 2017,2(11):1594-1601; and Su et al., Front. Microbiol. 2019; 10:1077. In oneor more embodiments, the platform is preferably portable.

Such a platform may allow for real-time data collection. The datacollected may include real-time changes in the magnetoresistance ratio(AMR) from each GMR sensor due to binding of a magnetic tag to thesensor through capture antibody-antigen-detection antibody complex. Thedata may be transmitted to any suitable device including, for example, asmartphone, a tablet, or a computer (laptop or desktop). For example,data transmission may be done wirelessly thorough Bluetooth to asmartphone, a tablet, and a laptop computer or through a USB connectionto a laptop or a desktop computer.

Exemplary components of hand-held detection platform include a GMRsensors that includes two ferromagnetic layers separated by one or moremetallic layers. Exemplary GMR sensors are described in Wu et al., ACSSens. 2017, 2(11):1594-1601; and Su et al., Front. Microbiol. 2019;10:1077. The surface of the GMR sensor may be functionalized so that thetarget antigen (e.g., M. hyopneumoniae or a specific protein of M.hyopneumoniae) specifically binds to the sensor. When a magnetic tagincluding, for example, a magnetic nanoparticle (MNP) binds to thetarget antigens (including, for example, via an antibody, as shown inFIG. 7(D)), the stray field generated by the MNPs results in theresistance change of the GMR sensors. This change may be measured, and,further, the change is proportional to the number of captured targetantigens.

Any suitable magnetic tag may be used. Exemplary magnetic tags includemagnetic nanoparticles (MNP) including superparamagnetic nanoparticlesor microbeads. An exemplary superparamagnetic microbead is furtherdescribed in Su et al., Front. Microbiol. 2019; 10:1077.

As used in the preceding description, the words “preferred” and“preferably” refer to embodiments that may afford certain benefits undercertain circumstances. However, other embodiments may also be preferredunder the same or different circumstances. Furthermore, an indicationthat one or more embodiments is preferred does not imply that otherembodiments are not useful and is not intended to exclude otherembodiments from the scope of the invention.

In the preceding description and following claims, the term “and/or”means one or all of the listed elements or a combination of any two ormore of the listed elements; unless otherwise specified, “a,” “an,”“the,” and “at least one” are used interchangeably and mean one or morethan one; and the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

As used herein, the terms “comprises,” “comprising,” and variationsthereof are to be construed as open ended—i.e., additional elements orsteps are optional and may or may not be present. In contrast, use ofterm “consisting of” is meant to be limiting: “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. As used herein, the term “consistingessentially of” is meant to include any elements listed after thephrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they materially affect the activity or action of thelisted elements.

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

In the preceding description, particular embodiments may be described inisolation for clarity. Reference throughout this specification to “oneembodiment,” “an embodiment,” “certain embodiments,” “one or moreembodiments,” or “some embodiments,” etc., means that a particularfeature, configuration, composition, or characteristic described inconnection with the embodiment is included in at least one embodiment ofthe disclosure. Thus, the appearances of such phrases in various placesthroughout this specification are not necessarily referring to the sameembodiment of the disclosure. Furthermore, the particular features,configurations, compositions, or characteristics may be combined in anysuitable manner in one or more embodiments. Thus, features described inthe context of one embodiment may be combined with features described inthe context of a different embodiment except where the features arenecessarily mutually exclusive.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

All reagents, starting materials, and solvents used in the followingexamples were purchased from commercial suppliers and were used withoutfurther purification unless otherwise indicated.

Example 1

Monoclonal antibodies were generated by standard hybridoma technique.(See FIG. 1 .) Eight-week old female BALB/c mice were immunizedsubcutaneously with a preparation of membrane proteins of M.hyopneumoniae strain 232. The membrane proteins of M. hyopneumoniae wereprepared by treating cells with 1% Tween 20 for 30 minutes at 37° C. andcollecting the soluble fraction. Tween 20 was removed from thepreparation before immunization using detergent removal resin. Mice wereimmunized three times, three weeks apart. Splenocytes from immunizedmice were fused with SP2/0 myeloma cells by standard techniques using50% polyethylene glycol 4000.

Hybridoma cultures were screened for secretion of antibodies to M.hyopneumoniae using direct ELISA on wells coated with total cell lysate.

Antibody cross reactivity to M hyorhinis and M. flocculare wasdetermined by ELISA and Western blotting methods.

A sandwich ELISA was developed to detect M. hyopneumoniae antigens byusing polyclonal rabbit anti-M. hyopneumoniae as a capture antibody andmonoclonal antibody (produced by a subclone) as the detection antibody.The polyclonal rabbit anti-M. hyopneumoniae was prepared by immunizingrabbits with same membrane protein preparation of M. hyopneumoniae usedfor monoclonal antibody.

Initial screening of hybridoma cells revealed 23 clones secretingantibodies to M. hyopneumoniae with absorbance in ELISA ranging from 0.5to 4.00. Of these, four clones were selected for cross reactivity studyand three clones that were specific to M. hyopneumoniae and which showedno cross reactivity to M. hyorhinis and M. flocculare antigens wereidentified. (See FIG. 2 and FIG. 3 )

Two of the hybridoma clones (2C9 and 4G11) produced antibodies whichexhibited an absorbance of greater than 1.00 by ELISA and whichdemonstrated reactivity to different proteins of M. hyopneumoniae byWestern blot were sub-cloned by limiting dilution and used for furthercharacterization, resulting in 2C9.B3 (which produces antibodiesreferred to herein as MAb 2C9.B3 or MAb 2) and 4G11.A3 (which producesantibodies also referred to herein as MAb 4G11.A3 or MAb 4).

Cross-reactivity analysis by both Western blot and ELISA confirmed bothMAb 2C9.B3 and MAb 4G11.A3 are specific to M. hyopneumoniae. Isotypingshowed both MAb 2C9.B3 and MAb 4G11.A3 are IgG1 isotype.

The monoclonal antibodies produced by 2C9.B3 and 4G11.A3 (MAb 2C9.B3 andMAb 4G11.A3) were used as the detection antibody in a sandwich ELISA todetect different field isolates of M. hyopneumoniae. Results are shownin FIG. 4 and FIG. 5 . Antibodies from one of the clones (MAb 2C9.B3)detected all ten M. hyopneumoniae isolates tested that were obtainedfrom different swine farms.

Example 2

This Example describes the development of an ELISA assay that mayaccurately detect the presence of M. hyopneumoniae at concentrations aslow as 1 μg/mL.

A sandwich ELISA assay (as described in Example 1) was used to test forlab-grown M. hyopneumoniae diluted in buffer or M. hyopneumoniaeisolated from swine lung tissue. Varying concentrations (2 μg/mL, 3μg/mL, 5 μg/mL, or 10 μg/mL) of the capture antibody were used. Varyingamounts (1% and 3%) bovine serum albumin (BSA) in blocking buffer (PBSand 1% normal goat serum) was tested. Inclusion of detergents (NP40 andTween 20) was also tested.

Anti-mouse IgG (R&D Systems, Minneapolis, Minn.) was used as theenzyme-linked secondary antibody.

Undiluted culture supernatant from hybridoma cultures was used as thedetection antibody.

Detection was performed using 3,3′,5,5′-Tetramethylbenzidine (TMB)substrate (Thermo Fisher Scientific, Waltham, Mass.). 2M HCl was used asa stop solution

Results were measured using endpoint absorption at 450 nm with a delayof 100 milliseconds. The sensitivity and specificity of MAb 2 and MAb 4were examined. Exemplary results are shown in FIG. 6A.

A capture antibody concentration of 3 μg/mL was found to be optimalamong those tested (2 μg/mL, 3 μg/mL, 5 μg/mL, and 10 μg/mL).

3% BSA in blocking buffer was found to provide less background signalrelative to 1% BSA in blocking buffer.

Use of a detergent was found to be preferred, and NP40 allowed for moresensitive detection than Tween 20.

M. hyopneumoniae was detectable at concentrations in a range of 1 μg/mLto 200 μg/mL.

Example 3

Total RNA was isolated from the hybridoma cells following themanufacturer's instructions. Total RNA was then reverse-transcribed intocDNA using either isotype-specific anti-sense primers or universalprimers following the technical manual of SMARTScribe ReverseTranscriptase (Takara Bio Inc., San Jose, Calif.). Antibody fragments ofheavy chain and light chain were amplified according to the standardoperating procedure (SOP) of rapid amplification of cDNA ends (RACE)(GenScript Biotech Corp., Piscataway, N.J.). Amplified antibodyfragments were cloned into a standard cloning vector separately. ColonyPCR was performed to screen for clones with inserts of correct sizes.The consensus sequence was provided.

Example 4

This Example describes the planned development of a giantmagnetoresistance (GMR)-based portable diagnostic immunoassay platformfor pen-side detection of M. hyopneumoniae directly from swinerespiratory samples. An exemplary system that may be used is shown inFIG. 7 . This portable testing platform would allow the antibodiesdescribed herein to be used for pen-side testing, providing the swineindustry with a more effective tool for M. hyopneumoniae screening andmonitoring.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Sequence Listing Free Text MAb 2C9.B3 heavy chain DNA sequenceSEQ ID NO: 1 ATGGCTGTCT TGGGGCTGCT CTTCTGCCTGGTGACATTCC CAAGCTGTGT CCTATCCCAG GTGCAGCTGA AGCAGTCAGG ACCTGGCCTAGTGCAGCCCT CACAGAGCCT GTCCATCACC TGCACAGTCT CTGGTTTCTC ATTAACTACCTATGCTATAC ACTGGGTTCG CCAGTCTCCA GGAAAGGGTC TAGAGTGGCT GGGAGTGATATGGAGTGGTG GAAACACAGA CTATAATGCA GCTTTCATAT CCAGACTGAG CATCACTAAGGACAATTCCA AGAGCCAAGT TTTCTTTAAA ATGAACAGTC TGCAACCTAA TGACACAGCCATATATTATT GTGCCAGAGA GGCCTCTACT ATGATAACGA CGGACTACTG GGGCCAAGGCACCACTCTCA CAGTCTCCTC A Signal sequence: 1-57 FR1: 58-147 CDR1: 148-162FR2: 163-204 CDR2: 205-252 FR3: 253-348 CDR3: 349-378 FR4: 379-411MAb 2C9.B3 heavy chain amino acid sequence SEQ ID NO: 2MAVLGLLFCL VTFPSCVLSQ VQLKQSGPGL VQPSQSLSIT CTVSGFSLTT YAIHWVRQSPGKGLEWLGVI WSGGNTDYNA AFISRLSITK DNSKSQVFFK MNSLQPNDTA IYYCAREASTMITTDYWGQG TTLTVSS Signal peptide: 1-19 FR1: 20-49 CDR1: 50-54FR2: 55-68 CDR2: 69-84 FR3: 85-116 CDR3: 117-126 FR4: 127-137MAb 2C9.B3 light chain DNA sequence SEQ ID NO: 3ATGGAGTCAC AGATTCAGGC ATTTGTATTC GTGTTTCTCT GGTTGTCTGG TGTTGACGGAGACATTGTGA TGACCCAGTC TCACAAATTC ATGTCCACAT CAGTAGGAGA TAGGGTCACCATCACCTGCA GGGCCAGTCA GGATGTGAGT ACTGCTGTAG CCTGGTATCA ACAAAAACCAGGCCAATCTC CTAAACTACT GATTTACTGG GCATCCATCC GGCACACTGG AGTCCCTGATCGCTTCACAG GCAGTGGATC TGGGACAGAT TATACTCTCA CCATCAGCAG TGTGCAGGCTGAAGACCTGG CACTTTATTA CTGTCAGCAA CATTATTTCA CTCCGCTCAC GTTCGGTGCTGGGACCAAGC TGGAGCTGAA A Signal sequence: 1-60 FR1: 61-129 CDR1: 130-162FR2: 163-207 CDR2: 208-228 FR3: 229-324 CDR3: 325-351 FR4: 352-381MAb 2C9.B3 light chain amino acid sequence SEQ ID NO: 4MESQIQAFVF VFLWLSGVDG DIVMTQSHKF MSTSVGDRVT ITCRASQDVS TAVAWYQQKPGQSPKLLIYW ASIRHTGVPD RFTGSGSGTD YTLTISSVQA EDLALYYCQQ HYFTPLTFGAGTKLELK Signal peptide: 1-20 FR1: 21-43 CDR1: 44-54 FR2: 55-69CDR2: 70-76 FR3: 77-108 CDR3: 109-117 FR4: 118-127MAb 4G11.A3 heavy chain DNA sequence SEQ ID NO: 5ATGGAATGGC CTTGGATCTT TCTCTTCCTC CTGTCAGTAA CTGAAGGTGT CCACTCCCAGGTTCAGCTGC AGCAGTCTGG GGCTGAGCTG GTGAGGCCTG GGTCCTCAGT GAAGATTTCCTGCAGGGCTT CTGGCTATTT ATTCAGTACC TTCTGGATAA ACTGGGTGAA GCAGAGGCCTGGACAGGGTC TTGAGTGGAT TGGACAGATT TATCCTGGAG ATGGTGATAC TAACTACAATGGAAAGTTCA GGGGTAAAGC CACACTGACT GCAGACAAAT CCTCCAGCAC AGCCTACATGCAGCTCAGCA GCCTAACATC TGAGGACTCT GCGGTCTATT TCTGTGCAAG AAGGGAAGTTTTGATCTATG ATGATTACTA CGATGCTCTG GACTACTGGG GTCAAGGAAC CTCAGTCACCGTCTCCTCA Signal sequence: 1-57 FR1: 58-147 CDR1: 148-162 FR2: 163-204CDR2: 205-255 FR3: 256-351 CDR3: 352-396 FR4: 397-429MAb 4G11.A3 heavy chain amino acid sequence SEQ ID NO: 6MEWPWIFLFL LSVTEGVHSQ VQLQQSGAEL VRPGSSVKIS CRASGYLEST FWINWVKQRPGQGLEWIGQI YPGDGDTNYN GKFRGKATLT ADKSSSTAYM QLSSLTSEDS AVYFCARREVLIYDDYYDAL DYWGQGTSVT VSS Signal peptide: 1-19 FR1: 20-49 CDR1: 50-54FR2: 55-68 CDR2: 69-85 FR3: 86-117 CDR3: 118-132 FR4: 133-143MAb 4G11.A3 light chain DNA sequence SEQ ID NO: 7ATGGATTTTC AAGTGCAGAT TTTCAGCTTC CTGCTAATCA GTGCCTCAGT CATAATATCCAGAGGACAAA TTGTTCTCAC CCAGTCTCCA GCAATCATGT CTGCATCTCC AGGGGAGAAGGTCACCATGA CCTGCAGTGC CAGCTCAAGT GTAAATTCCA TGCACTGGTA CCAACAAAAGTCAGGCACCT CCCCCAAAAG ATGGATTTAT GACACATCCA AATTGGCTTC TGGAGTCCCTGCTCGCTTCA GTGGCAGTGG GTCTGGGACC TCTTACTCTC TCACAATCAG CAACATGGAGGCTGAAGATG CTGCCACTTA TTACTGCCAG CAGTGGAGTA GTAACCCGCT CACGTTCGGTGCTGGGACCA AGCTGGACCT GAAA Signal sequence: 1-66 FR1: 67-135CDR1: 136-165 FR2: 166-210 CDR2: 211-231 FR3: 232-327 CDR3: 328-354FR4: 355-384 MAb 4G11.A3 light chain amino acid sequence SEQ ID NO: 8MDFQVQIFSF LLISASVIIS RGQIVLTQSP AIMSASPGEK VTMTCSASSS VNSMHWYQQKSGTSPKRWIY DTSKLASGVP ARFSGSGSGT SYSLTISNME AEDAATYYCQ QWSSNPLTFGAGTKLDLK Signal peptide: 1-22 FR1: 23-45 CDR1: 46-55 FR2: 56-70CDR2: 71-77 FR3: 78-109 CDR3: 110-118 FR4: 119-128

1. An antibody that binds M hyopneumoniae, wherein the antibodycomprises: a heavy chain variable region comprising one or morecomplementary determining regions (CDRs) of the heavy chain variableregions of MAb 2C9.B3, wherein the one or more MAb 2C9.B3 heavy chainCDR comprises: amino acids 50-54 of SEQ ID NO:2, amino acids 69-84 ofSEQ ID NO:2, or amino acids 117-126 of SEQ ID NO:2; a light chainvariable region comprising one or more CDRs of the light chain variableregions of MAb 2C9.B3, wherein the one or more MAb 2C9.B3 light chainCDR comprises: amino acids 44-54 of SEQ ID NO:4, amino acids 70-76 ofSEQ ID NO:4, or amino acids 109-117 of SEQ ID NO:4; or one or more MAb2C9.B3 heavy chain CDR and one or more MAb 2C9.B3 light chain CDR. 2.The antibody of claim 1, wherein the antibody comprises: a heavy chainvariable reason comprising: amino acids 50-54 of SEQ ID NO:2, aminoacids 69-84 of SEQ ID NO:2, and amino acids 117-126 of SEQ ID NO:2; anda light chain variable reason comprising: amino acids 44-54 of SEQ IDNO:4, amino acids 70-76 of SEQ ID NO:4, and amino acids 109-117 of SEQID NO:4.
 3. The antibody of claim 1, wherein the antibody is humanized.4. The antibody of claim 1, wherein the antibody comprises an antibodyfragment.
 5. The antibody of claim 1, wherein the antibody is an IgGantibody.
 6. The antibody of claim 1, wherein the antibody is amonoclonal antibody.
 7. The antibody of claim 1, wherein the antibody isa chimeric antibody.
 8. A method comprising using the antibody of claim1 in an in vitro or in vivo diagnostic or therapeutic method.
 9. Themethod of claim 8, wherein the method comprises detecting the presenceof M. hyopneumoniae in a subject.
 10. The method of claim 8, wherein themethod comprises detecting of M. hyopneumoniae in a sample obtained froma subject.
 11. The method of claim 8, wherein the method comprisesperforming a magnetic bioassay or enzyme-linked immunosorbent assay(ELISA).
 12. The method of claim 11, wherein the method comprisesperforming sandwich ELISA.
 13. The method of claim 8, wherein the methodcomprises detecting a change in the magnetoresistance ratio (AMR) from agiant magnetoresistance (GMR) sensor.
 14. The method of claim 13,wherein the method comprises transmitting the detected change in themagnetoresistance ratio (AMR) to a smartphone, a tablet, or a computer.15. An antibody that binds M. hyopneumoniae, wherein the antibodycomprises: a heavy chain variable region comprising one or morecomplementary determining regions (CDRs) of the heavy chain variableregions of MAb 4G11.A3, wherein the one or more MAb 4G11.A3 heavy chainCDR comprises: amino acids 50-54 of SEQ ID NO:6, amino acids 69-85 ofSEQ ID NO:6, or amino acids 118-132 of SEQ ID NO:6; or a light chainvariable region comprising one or more CDRs of the light chain variableregions of MAb 4G11.A3, wherein the one or more MAb 4G11.A3 light chainCDR comprises: amino acids 46-55 of SEQ ID NO:8, amino acids 71-77 ofSEQ ID NO:8, or amino acids 110-118 of SEQ ID NO:8; or one or more MAb4G11.A3 heavy chain CDR and one or more MAb 4G11.A3 light chain CDR. 16.The antibody of claim 15, wherein the antibody comprises: a heavy chainvariable region comprising: amino acids 50-54 of SEQ ID NO:6, aminoacids 69-85 of SEQ ID NO:6, and amino acids 118-132 of SEQ ID NO:6; anda light chain variable region comprising: amino acids 46-55 of SEQ IDNO:8, amino acids 71-77 of SEQ ID NO:8, and amino acids 110-118 of SEQID NO:8.
 17. The antibody of claim 15, wherein the antibody ishumanized.
 18. The antibody of claim 15, wherein the antibody comprisesan antibody fragment.
 19. The antibody of claim 15, wherein the antibodyis an IgG antibody.
 20. The antibody of claim 15, wherein the antibodyis a monoclonal antibody.
 21. The antibody of claim 15, wherein theantibody is a chimeric antibody.
 22. A method comprising using theantibody of claim 15 in an in vitro or in vivo diagnostic or therapeuticmethod.
 23. The method of claim 22, wherein the method comprisesdetecting the presence of M. hyopneumoniae in a subject.
 24. The methodof claim 22, wherein the method comprises detecting of M. hyopneumoniaein a sample obtained from a subject.
 25. The method of claim 22, whereinthe method comprises performing a magnetic bioassay or enzyme-linkedimmunosorbent assay (ELISA).
 26. The method of claim 25, wherein themethod comprises performing sandwich ELISA.
 27. The method of claim 22,wherein the method comprises detecting a change in the magnetoresistanceratio (AMR) from a giant magnetoresistance (GMR) sensor.
 28. The methodof claim 27, wherein the method comprises transmitting the detectedchange in the magnetoresistance ratio (AMR) to a smartphone, a tablet,or a computer.
 29. A composition comprising the antibody of claim
 1. 30.A kit or a device comprising the antibody of claim
 1. 31. A compositioncomprising the monoclonal antibody of claim
 15. 32. A kit or a devicecomprising the monoclonal antibody of claim 15.